This past fiscal year we had two major projects related to the above goals: 1) Amikacin exposure and susceptibility of macrolide-resistant Mycobacterium abscessus, in a patient cohort at NIH receiving long-term treatment: M. abscessus complex is associated with antibiotic resistance and poor treatment outcomes. We described within-patient changes in M. abscessus complex resistance to clarithromycin and amikacin. Patients with amikacin exposure and a >50 month interval between M. abscessus complex isolates were identified. Antimicrobial susceptibility testing was performed on first and last isolates by broth microdilution, and genetic markers of resistance were identified. Sixteen patients were identified with a median amikacin exposure of 2.3 years (range: 0.6-8.6). Fifteen patients also received macrolides (median: 7.2 years, range: 1.3-10.7). All initial isolates were resistant to clarithromycin (MIC8). Two patients had later susceptible isolates, which were of a different subspecies (M. massiliense) than the initial isolates (M. abscessus). All initial isolates were susceptible or intermediate resistant to amikacin, and only one patient had a resistant final isolate (MIC>64), accompanied by an AG mutation at position 1408 of the 16S rRNA. FEV1 decreased significantly over the study period, while smear quantity and the proportions of patients with elevated C-reactive protein or cavitary lesions all increased significantly. Despite prolonged, mostly inhaled amikacin exposure, development of amikacin resistance was uncommon in this patient population; however, disease progression continued. 2) Epidemiology of NTM in CF Pulmonary nontuberculous mycobacterial (NTM) disease represents a significant threat to cystic fibrosis (CF) patients, with an estimated annual prevalence of 12%. Prior studies report an increasing annual NTM prevalence in the general population, though similar trends in persons with CF have not been assessed. Objectives: This study aimed to identify the prevalence, geographic patterns, temporal trends and risk factors for NTM positivity by mycobacterial species among persons with CF throughout the United States. Using annualized CF Patient Registry (CFPR) data from 2010-2014, we identified patients with mycobacterial culture results to estimate annual and period prevalence of pathogenic NTM species by demographic and geographic factors. Regression models were used to estimate the annual percent change over time and risk factors for NTM isolation. Geographic patterns were described and mapped. Of 16,153 included persons with CF, 3,211 (20%) had a pathogenic NTM species isolated at least once over the 5-year period; 1,949 (61%) had Mycobacterium avium complex (MAC) and 1,249 (39%) M. abscessus. Period prevalence for MAC was 12% (CI:12-13%), for M. abscessus 8% (CI: 7-8%), and for other NTM species 4% (CI: 3.8-4.3%). Period prevalence for MAC was nearly three-times greater among patients 60 years old with a body mass index <19 (33% CI: 16-51%); this trend was not present for M. abscessus patients (4% CI: 0-11%). NTM prevalence showed a significant relative increase of 5% per year, from 11.0% in 2010 to 13.4% in 2014 (p=0.0008), although this varied by geographic area. For M. abscessus, the highest prevalence states were Hawaii (50%), Florida (17%) and Louisiana (16%), while for MAC it was Nevada (24%), Kansas (21%), and Hawaii and Arizona (both 20%). Study participants with either MAC or M. abscessus were significantly more likely to have been diagnosed with CF at an older age (p<0.0001), have a lower BMI (p<0.0001), higher forced expiratory volume in one second (FEV1) percent predicted (p<0.01), and fewer years on chronic macrolide therapy (p<0.0001). NTM remains highly prevalent among adults and children with CF in the U.S., with one in five affected, and appears to be increasing over time. Variation in prevalence exists by geographic region and by patient-level factors, including older age and receiving an initial CF diagnosis later in life. Routine screening for NTM, including mycobacterial speciation, especially in high-risk geographic areas, is critical for better understanding its epidemiology and changes in prevalence over time.